Momiji Embryonic Stem Cell System Enabling Live Imaging of Random X-Chromosome Inactivation In Vitro During Embryonic
Manami Koshiguchi1,2, Nao Yonezawa3, Yu Hatano3,4
1Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan.
Abstract:
In eutherian mammals, X-chromosome inactivation (XCI) equalizes X-linked gene dosage between XX female mammals and XY male mammals. Random XCI (rXCI) begins shortly after implantation, but its direct observation in vivo is difficult. In vitro differentiation of female embryonic stem cells (ESCs) provides a useful model to study rXCI, although conventional XX ESCs often lose one X chromosome during culture and/or differentiation, hindering accurate analysis. We developed the Momiji ESC (version 2) system, in which each X chromosome carries distinct fluorescent reporters and drug-resistance markers. Drug selection before differentiation prevents X-chromosome loss, enabling faithful rXCI modeling and long-term single-cell live imaging. This protocol uses spinning-disk confocal microscopy with Z-stack acquisition, rapid multi-tile imaging, and low-phototoxicity time-lapse observation to monitor rXCI onset and progression for up to 7 days. The Momiji ESC system offers a robust platform for visualizing and analyzing XCI dynamics in vitro.
